EP2612904B1

EP2612904B1 - Anticaries compositions and probiotics/prebiotics

Info

Publication number: EP2612904B1
Application number: EP11781828.6A
Authority: EP
Inventors: Alejandro Mira Obrador
Original assignee: Centro Superior de Investigacion en Salud Publica CSISP
Current assignee: Centro Superior de Investigacion en Salud Publica CSISP
Priority date: 2010-08-31
Filing date: 2011-08-31
Publication date: 2016-09-21
Anticipated expiration: 2031-08-31
Also published as: AU2011298235B2; JP2013543374A; CA2808793A1; AU2011298235A1; CN103119153A; MX343566B; US20140147426A1; RU2013114313A; WO2012028759A2; EP2612904A2; CA2808793C; BR112013004702A2; MX2013002247A; US9629883B2; JP5921546B2; KR20140001832A; IL224767A; ES2608577T3; CN103119153B; WO2012028759A3

Description

FIELD OF THE INVENTION

The present invention belongs to the field of human health; more specifically, the field of bucco-dental health.

BACKGROUND

The human oral cavity is inhabited by hundreds of bacterial species, most of which are commensal species, necessary to maintain equilibrium in the oral ecosystem. However, some of them play a key role in the development of oral diseases, primarily dental caries and periodontal disease (1). Oral diseases begin with the growth of dental plaque, a biofilm formed by the accumulation of bacteria jointly with glycoproteins from human saliva and polysaccharides secreted by microbes (2). The subgingival plaque, located in the neutral or alkaline subgingival pocket, is typically inhabited by Gram-negative anaerobes and is responsible for the development of gingivitis and periodontitis. The supragingival dental plaque is formed on the surface of the tooth and includes acidogenic and acidophilic bacteria, which, upon fermenting the sugars ingested in the diet, produce acid and lower the pH. When the pH is too acidic (generally with a value less than 5.5), the tooth enamel is de-mineralised and destroyed, and, therefore, these bacteria are responsible for dental caries, which are considered to be the most widespread infectious disease in the world, affecting over 80% of the human population (3). A bad oral health may be associated with other pathologies, such as, for example, stomach ulcers, stomach cancer or cardiovascular diseases, amongst others.
One of the main reasons why, as of today, oral pathogens have still not been eradicated is the difficulty involved in studying microbial communities that inhabit the oral cavity, since, on the one hand, the complexity of the ecosystem (several hundreds of species have been detected, with numerous levels of interaction) makes it difficult to detect the potential pathogenic species (4); moreover, no single etiological agent may be identified, as in classic diseases, following Koch's postulates. This fact has been clearly demonstrated in periodontal disease, where there are at least 3 bacterial species belonging to very different taxonomic groups (the so-called "red complex" of periodontal pathogens) which have been associated with the development and progression of periodontal disease (5). On the other hand, a large proportion of oral bacteria cannot be cultured (6) and, therefore, traditional microbiological methods give an incomplete image of the natural communities that inhabit dental plaque. However, the current development of metagenomic techniques and Next-Generation Sequencing technologies allows for the study of the bacterial community as a whole, by analysing the total DNA of complex microbial samples (Metagenome) without the need to culture the bacteria themselves.
In this regard, pioneering studies in metagenomics have focused on the intestinal ecosystem primarily through a shot-gun approach, wherein the DNA is cloned in small-size plasmids, followed by traditional Sanger-type sequencing (7, 8). More recent approaches include sequencing of the ends of large-size fosmids (9) and use of the "Illumina" sequencing technology, which provides a high coverage of short sequences (10). Studies of the microbiota of the oral cavity, as well as of other human body habitats, such as the skin, the vagina or the respiratory tract, have focused on the sequencing of ribosomal RNA amplicons (11, 12). These studies have provided a substantial improvement of our knowledge about these bacterial communities as compared to prior research based on cultures, but estimates of microbial diversity are hindered by the biases in PCR amplification (i.e. PCR only detects the bacteria that are most similar to those already known, and on the basis of which the amplification primers are used, giving an incomplete image of the diversity present), the cloning bias (a large number of genes are not cloned because they are toxic for the host bacteria and, therefore, this method does not allow for the study of the entire genetic reservoir of the sample) and a low sequence length (the sequences in the Illumina technology have only between 35-70 nucleotides, which in most cases makes a reliable taxonomic or functional assignment impossible), together with the fact that, as mentioned above, a large proportion of oral bacteria cannot be cultured.
In order to resolve the aforementioned problems, the present invention discloses the obtainment of the metagenome of dental plaque by the direct sequencing of metagenomic DNA, using 454-pyrosequencing, thereby eliminating the potential biases imposed by cloning and PCR techniques, and, furthermore, providing access to the entire genetic repertoire of the oral bacterial community under different health conditions, as well as the possibility to analyse which bacterial species amongst those found in the metagenome obtained may be associated with a good oral health, since those individuals who had never suffered from caries exhibit a different bacterial flora than those individuals who had suffered or currently suffer from it. By means of the oral metagenome obtained in the present invention, it is possible to direct the isolation, culture and identification of strains with anti-cariogenic activity from the conglomerate of bacteria in the buccal cavity sample; specifically, the supragingival plaque of individuals who have never suffered from caries, i.e. those strains capable of inhibiting the growth of cariogenic bacteria.
Another strategy disclosed in the present invention is the obtainment of a metagenomic library of fosmids (long DNA inserts, approximately 35-45 Kb) from the dental plaque of individuals who have never suffered from caries. By obtaining said fosmid library, it is possible to isolate and identify the bioactive anti-cariogenic peptides synthesised by the bacteria present in the oral cavity of individuals who have never suffered from caries. In this regard, given that, in the state of the art, Streptococcus mutans has been shown to be the main causal agent of caries (13), it is not surprising that most strategies against this disease have been aimed against said microorganism. These strategies have included the development of vaccines using known surface antigens, passive immunisation strategies that may neutralise the bacteria, the co-aggregation of S. mutans with probiotic strains and the use of inhibitory proteins specific to S. mutans, amongst others (14).
Other different strategies have been those disclosed in different patent documents, which propose the use of different bacterial strains, preferably S. mutans, that produce a lower concentration of acid (15), or the use of the same resources, for example, the nutrients, by pathogenic strains and non-pathogenic strains, continuously supplying high concentrations of non-pathogenic bacteria, which results in the displacement of the pathogenic bacteria, provided that they share the same resource (16), or even a lower adherence of cariogenic bacterial strains to the tooth (17). On the contrary, the bioactive strains and peptides disclosed in the present invention have antibiotic activity, preferably anti-cariogenic activity, on their own, against caries-producing microorganisms. On the other hand, patent WO20040072093 (18) discloses a number of anti-microbial agents that are active primarily against Gram-negative microorganisms, but the main causal agents of caries, S. mutans and S. sobrinus, are Gram-positive microorganisms. Moreover, the S. mitis and S. oralis isolates that produce the anti-microbial peptides disclosed in WO20040072093 (18) have been isolated from the throat of patients with cystic fibrosis, not from the mouth of people without caries, as in the case of the peptides and/or strains of the invention. Similarly, the therapeutic use of said peptides is aimed at the treatment of respiratory tract diseases and not of caries, as in the case of the bioactive peptides disclosed in the present invention.
In this regard, the main technical characteristics that make the bacterial strains isolated and disclosed in the present invention different from the rest of strains disclosed in the state of the art are that they may be cultured by means of conventional microbiological techniques; that they present inhibitory activity against organisms that produce infectious diseases of the buccal cavity, preferably caries, without the need to be genetically modified; and that they have been isolated from individuals who have never suffered from caries. Consequently, both the anti-cariogenic bacteria themselves and the bioactive anti-cariogenic compounds, preferably peptides, disclosed in the present invention may be used as probiotic and/or prebiotic compositions as such, or as a part of different pharmaceutical compositions used for the treatment of infections of the buccal cavity, such as, for example, caries, periodontitis, etc., or even as functional foods. Moreover, the present invention also discloses a method for the prevention and/or treatment of infectious diseases of the buccal cavity, preferably caries, that comprises the administration of a pharmaceutically effective quantity of at least one of the strains and/or at least one of the anti-microbial compounds, preferably peptides, described above, or of the probiotic or pharmaceutical composition or functional foods that comprise at least one of the strains and/or at least one of the compounds, preferably peptides, of the invention.

DESCRIPTION OF THE INVENTION

Brief description of the invention

The existing difficulty in the state of the art in identifying bacterial strains that directly inhibit the growth of pathogenic germs related to the onset of buccal cavity diseases is thus caused by the large quantity of bacterial species in said cavity; consequently, the difficulty involved in isolating, amongst all of them, strains that directly inhibit the growth of pathogenic species, many of which are non-culturable species, has made this problem hard to solve thus far.
The present invention resolves this problem by creating the metagenome of the buccal cavity of individuals who have never suffered from caries. The creation of said metagenome, using massive sequencing, preferably pyrosequencing, of the DNA present in the samples taken from the buccal cavity of said individuals who have never suffered from caries, makes it possible to identify the genera and species of the bacteria that are most frequent in the bacterial population present in the buccal cavity of said individuals. This quantification of the frequency of each bacteria in the sample had not been possible, thus far, using culture, cloning or PCR techniques, since these techniques only identify part of the bacteria and the proportions of those that are identified is biased due to the methodology itself (primarily due to the preferable culture, cloning or amplification of certain species, respectively).
In principle, the invention has been based on human beings, but could be applied to any superior mammal, especially pets or livestock, or even wild fauna. It would be sufficient to determine the characteristic metagenome of each species, in individuals who have never suffered from caries, as a representative disease of the typical buccal cavity diseases. Once the bacterial strains that are most frequent in the buccal cavity of healthy individuals have been identified from the data obtained from the metagenome, the following step of the present invention consists of culturing the samples obtained from the buccal cavity of these individuals, in the favourable culture media and under favourable conditions, such that the most frequent genera and species identified in the metagenome of the mammalian species under study may develop.
In the present invention, direct inhibitory capacity is defined as the capacity to completely inhibit growth, by creating inhibition haloes in lawn cultures of said pathogenic species, due to their antibiotic action, without ruling out the fact that, in addition to said inhibition caused by their antibiotic effect, the strains and compounds may exert their anti-microbial effect, preferably anti-cariogenic effect, by hindering the cariogenic action by other routes, such as modifying the optimal pH for the growth of said cariogenic strains, hindering their adherence to the teeth, etc.
To this end, the samples obtained from the buccal cavity of healthy individuals were lysed, the DNA thereof was extracted, fosmids were constructed with said fragments and cloned in a host cell that may be cultured and assayed in cultures of cariogenic species, in order to observe whether inhibition haloes against the growth of the pathogenic cariogenic species are produced.
It must be noted that, although the isolated inhibitory strains have been obtained from samples of the buccal cavity and are active against pathogenic caries-producing (cariogenic) bacterial species, given their inhibitory capacity against the growth of pathogenic bacteria that preferably inhabit the buccal cavity, in principle the bacterial strains could be found in other parts of the body and produce or be associated with other diseases. For this reason, an object of the present invention is the use of the strains as medicaments, particularly as anti-microbial agents and, more specifically, as anti-bacterial agents.
Therefore, the present invention discloses the isolation of culturable bacterial strains with an inhibitory capacity against the growth of pathogenic microorganisms involved in the onset of buccal cavity diseases. Throughout the present invention, the onset of caries has been taken as a representative disease of diseases typical of the buccal cavity, but the invention may be applied to any infectious disease attributable to pathogenic microorganisms of the buccal cavity. For this reason, the present invention preferably focuses on the isolation of bacterial strains with an inhibitory capacity against the growth of pathogenic microorganisms, particularly those involved in the onset of caries.
The process for isolating culturable bacterial strains with anti-cariogenic capacity is based on obtaining the oral metagenome of individuals who have never suffered from caries, in order to determine which type of bacteria said individuals present most frequently in their buccal cavity and analyse which of them are associated with a good oral health, by inhibiting the growth of cariogenic bacteria. Said process has made it possible to isolate, characterise, culture and deposit different strains with anti-cariogenic activity in the Spanish Type Culture Collection (CECT): CECT 7746, CECT 7747, CECT 7773 and CECT 7775. By means of sequence homology analysis, it was concluded that the four of the strains belonged to the same bacterial genus: Streptococcus; therefore, in addition to their functionality (anti-cariogenic activity) and the process for the obtainment thereof, said strains share a structural and taxonomic similarity, since they belong, as previously mentioned, to the same bacterial genus, Streptococcus.
Another aspect of the present invention discloses different specific culturable bacterial strains, CECT 7746, CECT 7747, CECT 7773 and CECT 7775, isolated from individuals with an excellent oral health who have never suffered from caries, characterised in that they present inhibitory activity against organisms that produce infectious diseases of the buccal cavity, preferably against caries-producing microorganisms. From the metagenome of the bacteria present in the dental plaque of individuals who had never suffered from caries, the genera and species of the bacteria that appeared most frequently in healthy individuals who had never suffered from caries were identified by homology with the existing bacterial DNA libraries. The bacteria that appeared most frequently in individuals without caries and which appeared to be absent or with a very low frequency in individuals with caries belonged to one of the following genera: Rothia, Globicatella, Johnsonella, Kingella, Cardiobacterium, Phocoenobacter, Mannheimia, Haemophilus, Neisseria, Streptococcus and Aggregatibacter; the genus Streptococcus being amongst the most abundant. In this regard, the preferred bacterial strains of the invention are strains CECT 7746, CECT 7747, CECT 7773 and CECT 7775, all of them belonging to the genus Streptococcus.
Moreover, the invention discloses solid, powdery (for direct intake or in solution) or pasty compositions for buccal hygiene, such as toothpaste, chewing gum, candy, bars, etc., or liquid mouthwash solutions, such as collutories, syrups, drinks, etc., or probiotic and/or prebiotic food compositions the composition whereof comprises either the strains of the invention, with inhibitory activity against organisms that produce infectious diseases of the buccal cavity, preferably caries-producing microorganisms. In a preferred embodiment of the invention, the strains of the invention are added to compositions that present anti-microbial activity against the flora of the buccal cavity and which may be commercially found, such as collutories of the Listerine^® type, said collutories showing an improved inhibitory effect against organisms that produce infectious diseases of the buccal cavity, preferably caries-producing microorganisms, when the strains and/or peptides of the invention are added to the composition thereof.
A preferred embodiment of the invention are probiotics/prebiotics or functional foods, the composition whereof comprises the strains of the invention, with inhibitory activity against organisms that produce infectious diseases of the buccal cavity, preferably caries-producing microorganisms. The concept of probiotics or functional foods includes, without being limited thereto: dairy products, such as yogurts, for example, juices, solid foods, such as sweets, for example, as well as teas, herbalist and parapharmacy products, such as vitamin complexes, with nutritional supplements, etc.
For purposes of the present invention, the following terms are explained:

Infectious disease of the buccal cavity: for purposes of the present invention, the infectious diseases of the buccal cavity are preferably caries, periodontitis, gingivitis and halitosis.
Probiotics: for purposes of the present invention, the term probiotic refers to the use of live microorganisms that are added to foods (milk, yogurts, etc.), dietary supplements (in the form of capsules, tablets, pills, powder, etc.) or others, which remain active and exert their physiological effects on the subject that ingests the food or similar product containing said probiotic. Ingested in sufficient quantities they have beneficial effects, in this case, on buccal health.
Prebiotics: for purposes of the present invention, the term prebiotic refers to the use of substances that are added to foods, chewing gum, dietary supplements or others, which exert an effect on the composition of the oral microbiota, favouring the establishment of bacteria that are beneficial for oral health and/or hindering the establishment of pathogenic bacteria.
Metagenome: represents the genomes of all the bacteria that are present in a sample, an individual or an ecosystem, etc.
Microbiome: is the set of microbes or bacteria that co-exist with human beings.
Bioactive anti-microbial compounds: are compounds such as biologically active peptides, proteins, antibiotics, pigments, etc. that are found in vertebrates and invertebrates and act as natural antibiotics, being a part of the innate immune response. Some of these compounds, for example peptides, are produced by human beings, such as, for example, defensins and cathelicidins, amongst others. They are active against bacteria, fungi and cloistered viruses.
Bacteriocins: are biologically active peptides secreted by bacteria that have bactericidal properties against other species that are closely related to the producing strain, or against strains that are phylogenetically distant from the producing strain.
Phosmids: circular DNA fragments that may be easily introduced into the host cells, generally bacterial cells, and transport bacterial or human DNA fragments.
Functional foods: are defined as those foods that are prepared not only for their nutritional characteristics, but also to fulfil a specific function, such as improving health or reducing the risk of contracting diseases. To this end, biologically active compounds, such as minerals, vitamins, fatty acids, bacteria with beneficial effects, dietary fibre and antioxidants, etc., are added thereto.
Culturable bacterial strains: culturable bacterial strains are considered to be those that grow in pure culture and keep growing in a stable manner, in an artificial laboratory culture medium under standard aerobiosis or anaerobiosis conditions.

Description of the figures

Figure 1 . A. Photograph of a Petri dish, which shows the initial screening of the clones of E. coli that contain the fosmids from DNA from the dental plaque of individuals without caries which produce inhibition haloes on a lawn culture of S. mutans. B. Photograph of a Petri dish, which shows the confirmation screening of the clones of E. coli that contain the fosmids from DNA from the dental plaque of individuals without caries that had produced inhibition haloes on a lawn culture of S. mutans.
Figure 2 . Photographs of Petri dishes that demonstrate the inhibition of the growth of lawn cultures of S. mutans in the presence of the isolates of the CECT 7746 (A), CECT 7747 (B), CECT 7773 (C), and CECT 7775 (D) strains disclosed in the invention.
Figure 3 . Photographs of Petri dishes that demonstrate the inhibition of the growth of lawn cultures of S. sobrinus in the presence of the isolates of the CECT 7746 (A), CECT 7747 (B) and CECT 7775 (C) strains disclosed in the invention.
Figure 4 . Growth curves of the S. mutans cariogenic bacteria in BHI culture medium in the presence of the supernatants, concentrated 10 times and isolated as a function of their molecular weight, obtained from cultures of strains CECT 7746 (A) and CECT 7747 (B) in the stationary phase. The data, taken every 15 minutes for 20 h, show the mean of 4 experiments. The line marked as antb represents the treatment with the antibiotic chloramphenicol (positive control). The X-axis of the graph shows the time, expressed in hours, and the Y-axis shows the optical density (OD) of the bacterial cultures.
Figure 5 . Growth curves of the S. mutans cariogenic bacteria in BHI culture medium in the presence of the supernatants, concentrated 10 times and isolated as a function of their molecular weight, obtained from cultures of strain CECT 7746 in the stationary phase (est) and the exponential phase (EXP). The data, taken every 15 minutes for 24 h, show the mean of 4 experiments. The line marked as clorf represents the treatment with the antibiotic chloramphenicol (positive control). The X-axis of the graph shows the time, expressed in hours, and the Y-axis shows the optical density (OD) of the bacterial cultures.
Figure 6 . Growth curves of the S. mutans cariogenic bacteria in BHI culture medium in the presence of the supernatant, concentrated 10 times and smaller than 3 kDa, obtained from cultures of strains CECT 7746 (A) and CECT 7747 (B), subjected to a treatment at 100ºC for 10 minutes. The data, taken every 15 minutes for 24 h, show the mean of 4 experiments. The X-axis of the graph shows the time, expressed in hours, and the Y-axis shows the optical density (OD) of the bacterial cultures.
Figure 7 . Photographs of Petri dishes that demonstrate the inhibition of the growth of lawn cultures of S. mutans in the presence of the supernatants of cultures of strains CECT 7746 (shown as 46 in the photograph) and CECT 7747 (shown as 47 in the photograph) under aerobiosis and anaerobiosis conditions.
Figure 8 . Concentration of lactic acid, expressed in mM, produced by the biofilm from the culture of human saliva in an artificial tooth model under aerobiosis and anaerobiosis conditions, whereto bacterial strains CECT 7746 and CECT 7747, or their respective supernatants, have been added. For more details, see Example 15. The negative control used was strain C7.1, which is an isolate belonging to the species of the Streptococcus mitis/oralis/infantis group, obtained from an individual without caries, but which does not produce inhibition of the growth of cariogenic species.

Detailed description of the invention

An object of the present invention is a culturable anti-microbial bacterial strain selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775. In a preferred embodiment, the anti-microbial bacterial strains disclosed in the invention present inhibitory activity against the growth of organisms that produce infectious diseases of the buccal cavity, preferably, caries-producing organisms. In a preferred embodiment, the strains of the invention are characterised in that, in addition to competitively growing to occupy the tooth, they are capable of producing inhibitory substances against the growth of cariogenic bacteria.
Another object of the present invention relates to the culturable anti-microbial bacterial strains selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, for use as a medicament.
Another object of the present invention relates to the use of at least one of the culturable anti-microbial bacterial strains selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, in the manufacturing of a medicament.
Another object of the present invention relates to a culturable anti-microbial bacterial strain selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, for use as an anti-microbial agent, preferably an anti-bacterial agent.
Another object of the present invention relates to the use of at least one of the culturable anti-microbial bacterial strains selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, in the manufacturing of an anti-microbial composition, preferably an anti-bacterial composition Another object of the present invention relates to a culturable anti-microbial bacterial strain selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, for use in the treatment of infectious diseases of the buccal cavity, preferably the treatment of caries.
Another object of the present invention relates to the use of at least one of the culturable anti-microbial bacterial strains selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, in the preparation of a composition designed for the treatment of infectious diseases of the buccal cavity, preferably the treatment of caries.
Another object of the present invention relates to the culturable anti-microbial bacterial strains selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, for use as a probiotic or functional food designed to improve buccal health, preferably to prevent caries.
Another object disclosed in the present invention relates to the use of at least one of the culturable anti-microbial bacterial strains selected from any of the following: CECT 7746, CECT 7747, CECT 7773 and CECT 7775, or a combination thereof, in the preparation of a probiotic or a functional food designed to improve buccal health, preferably to prevent caries.
Another object disclosed in the present invention relates to a probiotic/prebiotic composition or functional food that comprises at least one culturable anti-microbial strain, as mentioned throughout the present invention, as well as the anti-cariogenic substances, compounds or molecules secreted by said strains.
Another object disclosed in the present invention relates to a medical-pharmaceutical composition or a composition designed for buccal health that comprises at least one culturable anti-microbial strain as described throughout the present invention or the anti-cariogenic substances, compounds or molecules secreted by said strains.
Another object disclosed in the present invention relates to a process for isolating culturable anti-microbial bacterial strains, preferably with inhibitory activity against the growth of organisms that produce infectious diseases of the buccal cavity and, more preferably, caries-producing organisms, characterised in that it comprises:

a) Obtaining samples from the supragingival dental plaque of individuals who have never suffered from caries.
b) Seeding the samples in the adequate media and under the adequate conditions so as to grow and isolate only those bacteria that are most frequent in individuals who have never suffered from caries, estimating the latter by means of pyrosequencing of the metagenome.
c) Culturing the isolated strains in a growth medium for cariogenic bacteria and selecting, after an appropriate culture time, those strains that present inhibition haloes against said growth.

In a preferred embodiment, the process described above is characterised in that, in step b), the bacteria that are most frequent in individuals who have never suffered from caries are estimated by means of pyrosequencing of the metagenome, a technique that makes it possible to estimate the proportions of each bacterial species. In another preferred embodiment, the process described above is characterised in that, in step c), bacteria belonging to the following genera are preferably selected: Streptococcus, Rothia, Neisseria, Globicatella, Johnsonelia, Haemophilus, Kingella, Cardiobacterium, Mannheimia, Phocoenobacter and Aggregatibacter. Specifically, the following strains are selected: CECT 7746, CECT 7747, CECT 7773 and CECT 7775. More specifically, bacteria belonging to the genus Streptococcus are selected, preferably those belonging to the following species: S. sanguis, S. oralis, S. mitis, S. infantis or new species that have not been described but belong to the Streptococcus subgroup that includes these four species. More specifically, at least one anti-microbial bacterial strain is selected from: CECT 7746, CECT 7747, CECT 7773 or CECT 7775.
Another object disclosed in the present invention relates to a method for the prevention and/or treatment of infectious diseases, preferably of the buccal cavity and, more preferably, caries, which comprises the administration of a quantity that is effective to inhibit the growth of the pathogenic microorganisms, preferably cariogenic microorganisms, habitually present in said cavity, of at least one of the culturable anti-microbial strains described in the present invention; or the probiotic/prebiotic composition or the functional foods described in the present invention which comprise said strains; or the medical-pharmaceutical composition or the composition for buccal health described in the present invention which comprise said strains.
Another object disclosed in the present invention relates to a method for the prevention and/or treatment of infectious diseases, preferably of the buccal cavity and, more preferably, caries, which comprises the administration of a quantity that is effective to inhibit the growth of the pathogenic microorganisms habitually present in said cavity, of at least one anti-microbial compound as described throughout the present invention; or the probiotic/prebiotic composition or the functional foods which comprise at least one of the anti-microbial compounds described in the present invention, or the medical-pharmaceutical composition or the composition for buccal health which comprises at least one of the anti-microbial compounds described in the present invention.

Deposit of microorganisms in accordance with the Budapest treaty

The microorganisms used in the present invention were deposited in the Spanish Type Culture Collection (CECT), located in the Research Building of the University of Valencia, Campus Burjassot, Burjassot 46100 (Valencia, Spain), with deposit nos.:

CECT 7746: bacterial strain of the genus Streptococcus deposited on 7 June 2010.
CECT 7747: bacterial strain of the genus Streptococcus deposited on 7 June 2010.
CECT 7773: bacterial strain of the genus Streptococcus deposited on 22 July 2010.
CECT 7774: bacterial strain of the genus Rothia deposited on 22 July 2010.
CECT 7775: bacterial strain of the genus Streptococcus deposited on 22 July 2010.

The purpose of the examples listed below is to illustrate the invention without limiting the scope thereof.

Example 1. Obtainment of the metagenome of supragingival dental plaque

In the first place, samples of the supragingival dental plaque were taken from volunteers who have never suffered from caries, and, for comparative purposes, similar samples were taken from volunteers who had previously suffered from caries and volunteers who suffer from caries and, moreover, present lesions in said caries, after signing the informed consent. The sampling process was approved by the Clinical Research Ethics Committee of the General Directorate of Public Health of the Valencian Regional Government (GSP-CSISP). The buccal health condition of each volunteer was evaluated by a dentist following the recommendations and the nomenclature of the Studies in Buccal Health of the World Health Organisation (WHO), and the samples were taken using sterile probes. The volunteers were asked not to brush their teeth for 24 hours prior to the taking of samples.
In order to study the microbial diversity in the dental plaque and obtain the metagenome thereof, the material collected from the plaque of the surfaces of all the teeth of each individual was mixed in order to subsequently lyse it and obtain the total DNA of each dental plaque. The DNA was extracted using the MasterPure™ Complete DNA and RNA Purification Kit (Epicentre Biotechnologies), following the manufacturer's instructions and adding a treatment with lysozyme (1 mg/ml at 37ºC for 30 minutes) during the lysis step. The DNA concentration was measured with NanoDrop (Thermo Scientific) and the samples chosen must preferably have a DNA concentration greater than 300 µg/ml and a total quantity of at least 5 µg (due to the sensitivity threshold of the equipment and the processes involved in the pyrosequencing). Moreover, the DNA samples were run through an agarose gel in order to verify the integrity of the genomic DNA extracted from the volunteers' dental plaques. Subsequently, the pyrosequencing of said extracted DNA was performed by means of the GS FLX-Titanium Chemistry sequencer (Roche). Pyrosequencing consists of the fragmentation of DNA into fragments of about 500-800 nucleotides by means of nitrogen under pressure, adding adapters at the ends which make it possible to anchor the DNA to spheres of less than one micrometre in diameter. The spheres are introduced into an specific oil that acts as a microreactor, in order to perform an emulsion PCR (emPCR), where the DNA integrated in each sphere is amplified.
Following an enrichment of the spheres that have amplified the DNA, the solution is placed on titanium plates in the GS FLX sequencer (Roche), where the pyrosequencing reaction takes place. This reaction consists of the transformation of each pyrophosphate molecule released by polymerase upon adding a nucleotide in a light beam, by means of a set of enzymes such as luciferase. This light beam is proportional to the number of nucleotides added and, in this manner, a high-sensitivity chamber translates the light pulses into the corresponding DNA sequence (19). The average length of said DNA was 425 pb. Those sequences artifactually replicated by means of the 454-pyrosequencing technique that appeared systematically were eliminated from the set of final data through the "454 Replicar Filter" (20), such that the number of reads of a given sequence was related only to the frequency thereof in the sample.

The quantity of human DNA in the metagenomes ranged between 0.5%-40% in the samples from the supragingival dental plaque (Table 1) and were identified using the human genome database by means of Megablast (21) and eliminated from the set of final data.

Table 1. Characteristics of the pyrosequenced oral samples and the metagenome thereof.

Sample¹	CAO Index²	No. of reads	% human DNA	Total Mbp	Contigs > 5kbp	Largest contig	16S reads³	Simpson Index⁴	Shannon Index⁴	Chao1 Index⁴
NOCA_01P	0	347927	40.59	77.54	13	12856	543	0.93	3.19	100 ± 24.6
NOCA_03P	0	347927	22.76	100.13	49	43857	374	0.91	2.94	92 ±
CA1_01P	8(1)	494659	2.23	203.71	657	46856	1160	0.94	3.21	120 ± 24.8
CA1_02P	6(4)	315892	2.74	129.85	154	15919	575	0.92	3.11	85.2 ± 9
CA_04P	25 (15)	402049	11.54	142.37	181	19835	663	0.89	2.89	74.4 ± 9.9
CA_06P	11 (8)	354192	10.83	123.27	47	51033	615	0.95	3.38	129.2 ± 41
CA_06_1.6	11 (8)	305820	66.97	37.52	0	3376	194	0.92	3.21	77 ± 13.3
CA_05_4.6	10(7)	291162	74.99	27.67	2	29784	130	0.88	2.82	55.3 ± 8.3

¹ "P" indicates supragingival dental plaque samples. The samples with a number code indicate the tooth wherefrom samples from the caries cavity have been extracted.
² Number of dental pieces with caries (C), absent (A) and obstructed (O). The number in parenthesis indicates the number of exposed caries in the patient.
³ Number of sequences of 16S rRNA detected in the metagenome and assigned by means of the RDP classifier.
⁴ Simpson, Shannon, and Chao1 diversity indices, performed at the genus level.

Subsequently, a mean of 425 pbs allowed for the functional assignment in a significant fraction of the metagenome (Table 2). Moreover, the assembly of said reads produced 1103 assemblies ["contigs"] greater than 5 kpb and 354 greater than 10 kpb. We obtained a mean of 129.5 Mpb of high-quality filtered sequences (greater than 100 pb and where over 90% of the nucleotides had a 99.99% accuracy: The probability that the nucleotide read by the pyrosequencer is correct; i.e., a 99.99% accuracy means that only 0.01% of the nucleotides are incorrect (sequencing errors)) for each of the 6 oral samples. In the two samples with caries lesions, about 70% of the sequences pertained to human DNA, and in this case an average of 32.5 Mpb of high-quality filtered reads were obtained.

Table 2. Functional assignment of the samples present in the oral metagenome on the basis of different classification systems

Sample	Dental health¹	Total reads	cdd (n) ^a	cd (%)	cog (n) ^b	cog (%)	Tfam (n)^c	Tfam (%)	seed (n)^d	seed (%)
NOCA_01P	h	204218	126729	62	108929	53	82457	40	111497	50
NOCA_03P	h	244881	116575	48	95327	39	74356	30	93391	38
CA1_01P	c	464594	321997	69	280652	60	214050	46	271868	59
CA1_02P	c	295072	182091	62	150966	51	118716	40	146161	55
CA_04P	ac	339503	192003	57	161384	48	126281	37	158887	48
CA_06P	ac	306740	182349	59	151524	49	119477	39	146032	47
CA_05_4.6	cav	70503	40999	58	31864	45	26245	37	29625	42
CA_06_1.6	cav	97722	54305	56	45440	46	35395	36	44552	46

¹ h: healthy individuals without caries; c: individuals with caries in the past; ac: individuals with active caries; cav: samples of caries lesions.
(n): absolute count.
(%): total percentage of reads in the sample that were assigned to a function.
^acdd : assignment to conserved domains analysed in the NCBI Conserved Domains database.
^bcog: assignment to sets of orthologous groups.
^cTfam: assignment to Tigr Fams.
^dseed : assignment to the Seed / MG-RAST subsystems.

Example 2. Construction of the fosmid metagenomic library of supragingival dental plaque

Using the samples of intact DNA extracted from the supragingival dental plaque of the healthy volunteers included in the study which had not been used for the pyrosequencing process, the metagenomic library of fosmids (inserts that have a length of preferably between 35-45 kb) of the dental plaque of said volunteers was performed, using, to this end, the EpiFOS™ Fosmid Library Production Kit (Epicentre Biotechnologies), following the instructions provided by the manufacturer. Briefly, in a preferred alternative of the process for constructing the metagenomic library of the invention, the fosmids are inserted into a host, preferably Escherichia coli. The library is prepared, as explained above, using the EpiFOS™ Fosmid Library Production Kit (Epicentre), following the manufacturer's instructions with some modifications, such as increasing the ligation time (12 hours in a bath at 20ºC), the use of the total DNA for the insertion, and not the DNA extracted from the pulse-field gel, slightly modifying the DNA extraction process such that the latter breaks as little as possible (use of cut pipette tips, avoiding the vortex, use of Centricom membranes (Millipore) to concentrate the DNA).
Insertion of the DNA into the E. coli host is performed by packaging the fosmids in lambda phage particles and subsequently infecting them in Epi300T1 R strains of E. coli. During the packaging, the ligation product is placed in contact with the viruses for 3 hours at 30ºC in 1 ml of phage buffer. The infection is performed at 37ºC for 30 minutes, by placing the virus particles in contact with the E. coli strain. A prior titration is performed in order to select the optimal concentration of colonies in a plate (sufficiently distant so as to be able to seed a single colony with the aid of a sterile stick), by culturing different dilutions of the mixture in LB-agar medium with chloramphenicol.
Subsequently, each colony is inoculated in a 96-well Elisa plate in liquid LB medium with chloramphenicol, where they will be allowed to grow once again prior to being stored. The clones are stored in 96-well Elisa-type plates (Nunc) at a temperature of -80ºC in 19% glycerol, in order to prevent the formation of ice crystals and maintain the integrity of the cells. The fosmids are frozen without being induced to multiple-copy in order to prevent recombination processes between them.
The different E. coli clones with the different fosmid inserts are then seeded in cultures of cariogenic bacteria, such as Streptococcus mutans or Streptococcus sobrinus. Those clones are selected which, in said cultures, present an inhibition halo around the seeding point (Figure 1). The clones obtained are identified by means of sequence homology of the DNA contained in each fosmid, on the basis of different available public sequence databases. In order to obtain the DNA sequence of each fosmid, the total DNA thereof is extracted by separating it from the vector DNA by means of midiprep kits from QIAGEN, and performing the direct pyrosequencing thereof. This is how the respective ORFs and, subsequently, the peptides encoded thereby, are obtained.

Example 3. Analysis of the diversity of the human oral Metagenome

Once the metagenome of the supragingival dental plaque from individuals with caries and from healthy individuals was obtained following the process described in the present invention, the diversity of said oral metagenome was analysed using three different techniques:

Taxonomic assignment by means of the analysis of the 16S rRNA: the 16S rRNA sequences were extracted from the reads obtained from each metagenome by means of similarity searches with BLASTN (26) against the RDP database (Ribosomal Database Project). The sequences with a size smaller than 200 pb were eliminated. The phylogenetic assignment of the sequences was performed using the RDP Classifier (27), with a confidence threshold of 80%.
Gene taxonomic assignment: the taxonomic assignment of all the ORFs was performed on the basis of the lowest common ancestor (LCA) algorithm, by means of the characteristics described in the MEGAN software (28). In order to obtain the LCA of each sequence, homology searches were performed using the BLASTx database against another customised database that includes non-eukaryotic sequences from the non-redundant NCBI database (NR). For each sequence read, only those results that showed a coincidence of at least 90% were considered in the obtainment of the LCA.
Taxonomic assignment of the reads (PhyMM): said taxonomic assignment is performed using PhymmBL (29), which combines the assignment of sequences by both homology and the composition of nucleotides; to this end, hidden Markov models are used. All the available complete genomes were obtained from the Human Oral Microbiome Database (HOMD) (30), as well as the NCBI database (RefSeq), which contain all the genomes of bacteria and archaea (March 2010), and were used to construct a local database designed to perform taxonomic construction models and homology searches by means of PhymmBL. In this analysis, we only used sequences greater than 200 pb to predict the taxonomic identification. Using said read length, the class-level accuracy of the search with PhymmBL has been estimated to be greater than 75%. All the taxonomic and functional results were analysed in a MySQL database for the subsequent analysis thereof.

The results obtained using these three methods show that a small number of 16S genes in directly sequenced metagenomes are sufficient to describe the main taxonomic groups present in the oral metagenome, without the biases associated with cloning or PCR techniques.
From the samples examined, interesting differences may be observed between healthy and ill individuals. The trend shown by the three methods was that the Bacilli and Gamma-proteobacteria taxonomic groups were the most common in healthy individuals, whereas typically anaerobic taxa, such as Clostridials and Bacteroidets, are more frequent in samples from ill subjects. The reads assigned to Beta-proteobacteria (primarily Neisserials) and phylum TM7 (as yet without a definite name and without any member having been cultured thus far) were present in a very low proportion in samples from ill individuals and, therefore, may be associated with healthy conditions.
Correspondence analysis between the metagenomes, based on the taxonomic assignment of the 16S rRNA reads, showed that the samples from individuals with bad oral health tended to group together, whereas different bacterial consortia may be found in healthy individuals. By means of the present metagenomic study, the invention demonstrates that the genera Streptococcus and Rothia, more preferably, the genus Streptococcus, are prevalent genera in subjects without caries. For this reason, when we select, from the supragingival plaque samples of individuals who had never suffered from caries, those that could have anti-cariogenic activity, the selection was aimed at searching (culture media, culture parameters, microscope morphology of the bacteria, morphology of the colonies, etc.) species belonging to said genera, Streptococcus and Rothia, more preferably, the genus Streptococcus.
One of the powerful applications of the LCA and PhymmBL approaches is that most of the reads with significant coincidences may be assigned to a taxonomic origin and, moreover, to a possible function. By relating the taxonomy and the function, it has been possible to predict the ecological or metabolic role that each bacterial group may play. Using the COG (Cluster of Orthologous Groups) functional classification system, it may be observed that the categories are not evenly distributed, and that certain bacterial groups are especially suited to perform certain functions. For example, a large proportion of genes involved in defence mechanisms (e.g. restriction endonucleases and drug discharge pumps) are encoded by Bacilli, which, jointly with the greater presence of Streptococci in people without caries, allowed us to predict that those bacteria could be potential producers of natural inhibitors of human pathogens in a possible replacement therapy strategy for the treatment of buccal infectious diseases.

Example 4. Analysis of the microbial richness and abundance present in the human oral metagenome

Initial studies based on traditional culture techniques and pioneering molecular works, including amplification and cloning of the 16S rRNA gene, predicted a diversity of about 500 different species in the oral cavity (6). The use of last-generation technologies (Next Generation Sequencing, NGS) gave estimates of between 4000 and 19000 operational taxonomic units (OTUs). OTUs are estimates of the number of species on the basis of the DNA sequences, which take into consideration the fact that sequences of the 16S rRNA gene with a similarity lower than a given threshold belong to different species. The threshold used is the standard for the 16S rRNA gene, a 97% sequence identity; thus, if the similarity is greater than 97%, it is considered to be of the same species, but, if it is lower than 97%, it is probably a different species. Longer pyrosequencing reads (250 pbs) in three healthy subjects estimated about 600 OTUs per person, and a recent project attempted to sequence 11447 amplicons with almost the full length of 16S rRNA amplicons using Sanger-type sequencing (22), reducing the estimates to less than 300 OTUs in 10 individuals.
Although our estimates of microbial diversity are closer to those obtained using Sanger-sequenced reads (6,22), the 16S rRNA reads extracted from our metagenomic data identified 186 new OTUs that had not been previously detected by PCR amplification. The rarefaction curves (the saturation in the number of species as the sampling stress increases) and different diversity indices, as described in Table 1 (specifically, the Shannon, Simpson, and Chao1 indices), based on 4254 rRNA reads, indicated an estimate of 73-120 genera for the dental plaque samples (Tables 1 and 2). Clear differences between the samples of volunteers with different health conditions were not observed in regards to the diversity, although the two samples with caries lesions tended to present a lower diversity.
An effective tool to quantify the presence of selected species in metagenomes is sequence recruitment. Those individual metagenomic reads with coincidences greater than a certain identity threshold against a reference bacterial genome are "recruited" to plot a graph that will vary in density depending on the abundance of that organism in the sample. If the mean nucleotide identity shown is greater than 94%, the recruitment has probably been performed against reads from the same species (23).
Upon comparing our metagenomes with the 1117 genomes available thus far, using the Nucmer and Promer v 3.06 algorithms, we have been able to estimate the abundance of these species in our samples. Surprisingly, bacteria related to Aggregatibacter and Streptococcus sanguis were amongst the most abundant in people without caries, which agrees with the greater PCR amplification frequency of these species in the oral cavity of healthy individuals. The genus Neisseria was also frequent in samples from healthy individuals. Moreover, the recruitment graphs indicate that a few taxa are normally dominant in each metagenome, which suggests that, although there is a great bacterial diversity in the oral cavity, a few taxa comprise most of the bacterial cells.

Example 5. Functional diversity in the oral ecosystem

In order to analyse the functional diversity of the organisms that are a part of the oral ecosystem of the individuals analysed in the present invention, all the metagenomic sequence reads obtained were compared to different databases: conserved domain database (CDD) (24), subsystem-based annotation system (SEED) and TigrFams profiles (25).
Correspondence analysis (CoA) of the samples on the basis of the functional assignment of the reads provided similar grouping patterns for the three functional classification systems (CDD, SEED and TigrFams). The samples from ill subjects (with caries) tended to group together, indicating that a similar group of functions were encoded in their metagenomes, and the samples from individuals who had never suffered from caries, jointly with one of the individuals who presented a low number thereof, are separately grouped. When comparing the functional assignment of the oral metagenomes against the intestinal microbiome of adult persons (8), the oral samples are grouped together, indicating that the intestine and the mouth are two different ecosystems in terms of the relative frequencies of the encoded functions. The present invention demonstrates that there are blocks of functions that are over-represented in the intestinal microbiome, whereas others are over-represented in the oral samples.
In the oral samples, the individuals are grouped on the basis of their health condition. From an applied standpoint, it is interesting to note that many functional categories are over-represented in the samples from individuals without caries. These include DNA capture genes involved in competition in Gram-positive bacteria, others involved in the phospholipid metabolism, fructose and mannose-induced phosphotransferase systems, the Streptococcus mga regulon, proteins involved in mixed acid fermentation, quorum-sensing genes and bacteriocin-type anti-bacterial peptides. Said bioactive compounds, bacteriocins, are potential anti-caries agents and, therefore, the present invention demonstrates that the dental plaque of individuals who have never suffered from caries is a genetic reservoir of new anti-microbial and potentially anti-cariogenic substances.

Example 6. Identification of anti-caries bacteria.

The existence of a small proportion of the adult human population that has never suffered from caries has led to suggest the presence of bacterial species with a potential antagonistic effect against cariogenic bacteria (23). Bacterial replacement of the pathogenic strains by innocuous isolates obtained from healthy individuals has satisfactorily proven to prevent pharyngeal infections and is the basis for probiotics designed to prevent infectious diseases in the intestine and other human ecosystems (31). Metagenomic recruitments of cariogenic bacteria against the oral microbiome of healthy subjects show a total absence of S. mutans and S sobrinus. Surprisingly, the lack of detection of cariogenic bacteria is accompanied by an intense recruitment of other species of Streptococcus (primarily those similar to S. sanguis), Aggregatibacterand Neisseria, which are the most abundant genera in these individuals.
Given the possibility that isolates of these dominant genera may be involved in antagonistic interactions with cariogenic bacteria, fresh samples of dental plaque were taken from 10 healthy individuals (including the 2 healthy individuals wherefrom the metagenomic sequences were obtained) and used to culture, under optimal growth conditions, species of Neisseria, Rothia and Streptococcus (specifically, in blood agar, chocolate agar, brucella agar and TSA culture media, under aerobiosis conditions and in anaerobic jars). Following a microscopic examination, diplococci and streptococci were selected (in order to maximise the possibility of finding species of Streptococcus, Rothia and Neisseria), and a set of 249 isolates was obtained.
Those that were able to grow in the same medium as S. mutans and S. sobrinus were transferred to lawn cultures in the presence of said cariogenic bacteria. This simple screening identified 16 strains with inhibition haloes (Figures 2 and 3). Using PCR techniques and sequencing of the 16S rRNA, most of said strains were identified as belonging to species of Streptococcus, showing a 96%-99% sequence identity with the S. oralis, S. mitis and S. sanguis species or other related species, and also with Rothia species, with a 100% sequence identity with the R. mucilaginosa species in the 16S gene. The strains that showed inhibition haloes against S. mutans and/or S. sobrinus were deposited in the CECT, being assigned numbers CECT 7746, CECT 7747, CECT 7773 and CECT 7775. As previously discussed, strains CECT 7746, CECT 7747, CECT 7773 and CECT 7775 belong to the same bacterial genus Streptococcus and, therefore, in addition to the method for obtaining them, share a structural and taxonomic similarity, since they belong to the same bacterial genus.
Specifically, on the basis of the 16S ribosomal gene sequence, said strains, which belong to the bacterial genus Streptococcus, are similar to the S. mitis (CECT 7746 and CECT 7775) and S. oralis (CECT 7747 and CECT 7773) species. Sequencing of the complete genome of strains CECT 7746 and 7747 reveals that they are new species of the genus Streptococcus (see Example 7), that they are sister strains despite coming from different individuals and, moreover, belong to the S. mitis/oralis/infantis cluster of species. The inhibition haloes against cultures of cariogenic species, S. mutans or S. sobrinus, of said strains deposited in the CECT may be observed in Figures 2 and 3, respectively.

Example 7. Characterisation of bacterial strains CECT 7746 and CECT 7747.

Characterisation of bacterial strains CECT 7746 and CECT 7747 was performed by means of different techniques. In the first place, the complete genome of these two strains was obtained by means of shot-gun (7, 8) and pair-ends pyrosequencing; the latter consists of breaking the DNA into fragments of about 3000 nucleotides and the sequencing of approximately 200 nucleotides from each end, such that the known distance between these two ends helps in the assembly of the sequences.
In order to obtain the complete genome for each of strains CECT 7746 and CECT 7747, we started from samples collected from each of the cultures of said strains; specifically, a one-fourth culture plate was used for the pyrosequencing experiments by means of the shot-gun system (7, 8), using the GS-FLX pyrosequencer from Roche (Titanium Chemistry), and another one-fourth culture plate was used for the pyrosequencing experiments using the pair-ends system. The sequence quantity obtained for each of the strains using both systems was:

Strain 7746
- Shot-gun type: 441.549 reads, with a total of 165,105,921 nucleotides
- Pair-ends type: 187.530 reads, with a total of 32,721,622 nucleotides
Strain 7747
- Shot-gun type: 28.021 reads, with a total of 5,711,998 nucleotides
- Pair-ends type: 305.826 reads, with a total of 51,501,510 nucleotides

The expected genome size for each of the strains was about 2.1 Mb. For strain CECT 7746, the size of the assemblies greater than 500 pb is 2,122,087 pb. In the case of strain CECT 7747, the size of the contigs greater than 500 pb is 1,953,989 pb.
The sequences were filtered and assembled using the Newbler software (Roche), adapted by the inventors with standard parameters, to obtain a total of 109 assemblies > 500 pb for strain CECT 7746 and of 51 contigs for strain CECT 7747. Subsequently, said genomes were automatically annotated, to obtain the complete sequence of the genome of said strains CECT 7746 and CECT 7747.
Once the complete genome of strains CECT 7746 and CECT 7747 was obtained, said isolates were taxonomically assigned on the basis of the phylogenetic trees obtained on the basis of the complete sequence of the 16S and 23S rRNA genes, which are the most widespread when preparing bacterial phylogenetic trees.
By linking together the sequences of the 16S and 23S rRNA genes, a single fragment greater than 4,000 nucleotides was obtained, which was aligned with the same fragment of the Streptococci species sequenced thus far. The sequences were aligned using the MAFFT free computer software, by aligning the 16S and 23S genes separately, and subsequently linking the alignment together. Afterward, the alignment is purified using the GBlocks free computer software in order to select the conserved informative positions. The tree is obtained using the RAxML programme, by the maximum verisimilitude method, with 500 repeats. The phylogenetic tree obtained showed that both strains are sister strains despite coming from different individuals and, moreover, belong to the S. mitis/oralis/infantis cluster of species, and the topology of the tree suggests that they are strains that belong to a new, non-described species.
In order to determine whether said strains belong to different species, the ANI (average nucleotide identity) was used. When the genomes of the sequenced strains are compared, the mean similarity between the homologous genes of the same species at the nucleotide level is greater than 95% (34, 35). In fact, taxonomists accept this 95% ANI value as the limit to separate bacterial isolates belonging to different species and as an alternative to the classic 70% threshold in the DNA-DNA hybridisation value (36). Using the J-species free computer software to determine the ANI values between the two sequenced strains of the invention, CECT 7746 and CECT 7747, and the rest of the Streptococci sequenced, it was demonstrated that they are two strains belonging to a new species that has not been described as yet. In the case of the bacterial strain of the invention CECT 7746, there is no strain with a similarity above the 95% threshold and, in the case of the bacterial strain of the invention CECT 7747, only another strain of those sequenced, Streptococcus M143, exceeds said threshold. Said strain M143 is a strain that, despite having a draft genome sequence, has not been taxonomically described as a species. The results used to calculate the ANIs were obtained by means of two different methodologies: Mummer and Blast (36), and both methodologies showed almost identical results.

Example 8. Inhibition assays of cariogenic bacteria, S. mutans, in the presence of the supernatants obtained from the cultures of the CECT 7746 and CECT 7747 strains disclosed in the invention.

The two strains of the invention, CECT 7746 and 7747, were grown in BHI culture medium at a temperature of 37ºC. Subsequently, the supernatants of the cultures were collected, in the exponential phase and the stationary phase, being filtered through a 0.2-micron filter, in order to eliminate any bacterial residue. Subsequently, said supernatants were filtered once again by means of centrifugation, using membranes with a pore size of 100, 10 and 3 kDa (Amicon, Millipore), as described in the previous examples shown in the present invention.
The fraction of the supernatants obtained from each of strains CECT 7746 and 7747, collected in the stationary phase of growth, which produced inhibition of the growth of bacterial cultures of S. mutans, was concentrated in the size fraction smaller than 3 kDa for both tested strains, CECT 7746 (Figure 4 A) and CECT 7747 (Figure 4 B). Said results show that the inhibitory substance synthesised by said strains, which presents specific bactericidal effect against cariogenic species, must be of a small size, preferably <3 kDa, as in the case of bacteriocins.
On the contrary, when the same experiment was performed with samples of the supernatants of cultures of the strains of the invention CECT 7746 and 7747, collected in the exponential phase of growth, no inhibition of the growth of bacterial cultures of S. mutans (Figure 5) was observed, which indicates that the inhibitory agent is only produced in the stationary phase of bacterial growth of the strains of the invention.
When the samples of the concentrated supernatant obtained in the stationary phase, and smaller than 3 kDa, were subjected to a temperature of 100ºC for 10 minutes, it was verified that the inhibitory activity of said supernatant on cultures of S. mutans was maintained and even increased (Figure 6). These results are consistent with the fact that the inhibitory agent is a bacteriocin and not another type of peptide, since small-size bacteriocins are extremely thermostable and even increase their anti-microbial effect, since they are better eluted in the medium after their aggregates are dissolved through thermal shock.
Subsequently, inhibition assays against cultures of S. mutans were performed with the supernatants obtained from the cultures of the bacterial strains of the invention CECT 7746 and CECT 7747, but changing the seeding order and the growth temperature of the cultures of said cariogenic bacteria, in order to verify whether said modifications had any effect on the inhibitory activity of the supernatants of the strains of the invention.
In the first place, culture plates were seeded with the S. mutans cariogenic bacteria and, after 24 h had elapsed, the strains of the invention were seeded in the same culture plate. After a time had elapsed, no inhibition haloes were observed. On the contrary, when culture plates were seeded with both strains at the same time, inhibition of the growth of S. mutans was observed, as we had previously shown. The greatest inhibition of the growth of cultures of S. mutans was observed when the strains of the invention, CECT 7746 and 7747, were seeded first and, 24 h later, the strains of S. mutans were seeded, which indicates that there is a greater concentration of the inhibitory agent or substance prior to the growth of the cariogenic bacteria, and that, moreover, the presence of said bacteria is not necessary to activate the production of the inhibitory agent by the strains of the invention.
Subsequently, inhibition experiments against the growth of cariogenic bacteria were performed in a solid medium, by first seeding the strains of the invention with a drop of the culture in a liquid medium in the stationary phase, followed by a tapestry culture of S. mutans at different temperatures: 30ºC, 33ºC and 36ºC. No inhibition of the growth of S. mutans was observed at a temperature of 30ºC, but inhibition was observed at 33ºC, being in fact greater than that obtained at 36ºC.

Example 9. Inhibition assays against cariogenic bacteria, S. mutans, cultured in the presence of the supernatants obtained from the cultures of the bacterial strains of the invention CECT 7746 and CECT 7747, under aerobiosis and anaerobiosis conditions.

In order to determine whether the inhibitory action of the strains of the invention against the growth of cariogenic bacteria was modified by an aerobic or anaerobic environment, inhibition experiments were performed in a solid BHI medium, by first seeding the strains of the invention with a drop of the culture in a liquid medium, in the stationary phase, in an anaerobic jar for 12 h, followed by a tapestry culture of S. mutans at 37ºC, or followed by seeding with one drop of the culture of S. mutans at 37ºC.
The results of both experiments showed that inhibition of the growth of S. mutans is much lower under anaerobic conditions, especially for strain CECT 7746 (Figure 7). Therefore, the results demonstrate that, for the strains of the invention, the inhibition exerted on cariogenic bacteria is more effective during the aerobic step of formation of the dental plaque, i.e. during the period of adherence and initial formation of the biofilm on the tooth.

Example 10. Anti-cariogenic effect of bacterial strains CECT 7746 and CECT 7747 and the supernatants thereof on the biofilm in an artificial tooth model.

In order to demonstrate the anti-cariogenic effect of the bacterial strains disclosed in the present invention, inhibition assays against the production of acid were performed with strains CECT 7746 and CECT 7747 on a biofilm in an artificial tooth model. Said experiments were performed on the Active Attachment biofilm model of the Academic Center for Dentistry Amsterdam (ACTA, Amsterdam). Said biofilm model was described by Exterkate RA et al. (37). Briefly, hydroxyapatite or glass discs are inoculated with human saliva from a volunteer with a high percentage of S. mutans (greater than 4%), with or without the presence of the probiotic strain, or the supernatant thereof. In the present assays, strains CECT 7746 and 7747, disclosed in the present invention, were tested, as was strain C7.1, an isolate belonging to species of the Streptococcus mitis/oralis/infantis group, obtained from an individual without caries, but which does not produce inhibition of the growth of cariogenic species and, therefore, acts as a negative control.
The human saliva is stored at -80 ºC. Probiotic strainsCECT 7746 and 7747 and control strain C7.1 are grown in BHI culture medium with sucrose for 12 hours, until a culture density of approximately 4 x 10⁸ cfu (Colony-Forming Units) is obtained. Subsequently, the saliva sample is mixed at 50% with the inoculum of the probiotic strains of the invention (CECT 7746 or 7747) or the inoculum of the control strain (C7.1), and applied onto the glass disc.
The biofilms are formed for 48 hours in modified artificial saliva medium (38) under aerobiosis and anaerobiosis, and, once formed, are incubated for 3 hours at a temperature of 37ºC in cysteine peptone water (Sigma-Aldrich, St Louis, USA) containing 0.2% glucose, in order to measure the production of acid. During this incubation period, the strains will produce acid, which is measured by means of a colorimetric reaction: Said biofilm is transferred to an Eppendorf tube and incubated at a temperature of 80ºC for 5 min in order to stop the bacterial metabolism. The quantity of L-lactic acid is enzymatically determined by means of a colorimetric assay using the Spectra Max M2 spectrophotometer (Molecular Devices, USA), following the protocol described by Pham LC et al. (38).
In order to analyse the inhibition of the production of acid by the supernatants, of the strains of the invention (CECT 7746 and 7747) as well as the control strain (C7.1), in the first place, the supernatants of the cultures of said bacterial strains were obtained. To this end, cultures of said strains were grown in BHI medium for 12 hours. Subsequently, the bacterial cells were eliminated by centrifugation and subsequent filtering through pores with a size of 0.2 microns. The medium is filtered through Amicon 100-, 10- and 3-kDa Ultra membranes (Millipore). The fraction smaller than 3 kDa is concentrated to half of its volume in a rotavapor and mixed at 50% with the saliva sample; subsequently, as in the case of the probiotics, the biofilm is formed for 48 hours and incubated for 3 hours in a Buffered Peptone Water culture medium (38) containing 0.2% glucose, so as to be able to measure the production of acid.
Each treatment is repeated in quadruplicate under aerobiosis and anaerobiosis conditions. The experimental groups analysed were:

1. Biofilms formed with saliva inoculum.
2. Biofilms formed with saliva inoculum + CECT 7746.
3. Biofilms formed with strain CECT 7746.
4. Biofilms formed with saliva inoculum + CECT 7747.
5. Biofilms formed with strain CECT 7747.
6. Biofilms formed with saliva inoculum + non-inhibitory Streptococcus strain (strain C7.1).
7. Biofilms formed with saliva inoculum + supernatant of strain CECT 7746, which contains the active inhibitory substance.
8. Biofilms formed with saliva inoculum + supernatant of strain CECT 7747, which contains the active inhibitory substance.
9. Biofilms formed with saliva inoculum + supernatant of the non-inhibitory strain (strain C7.1).
10. Biofilms formed with the non-inhibitory Streptococcus strain (strain C7.1).

The results are shown in Figure 8, and indicate that the monospecific biofilm formed solely by strains CECT 7746 (experimental group 3) or CECT 7747 (experimental group 5) produce a quantity of acid that is significantly lower than that of saliva (experimental group 1). Taking human saliva as the reference value (experimental group 1), the supernatants of strain CECT 7747 (experimental group 8) significantly reduced the production of acid, under both aerobiosis and anaerobiosis, whereas the supernatant of strain CECT 7746 (experimental group 7) reduced the quantity of acid produced by the biofilm only under anaerobiosis conditions. The addition of strain CECT 7747 to the biofilm reduced the production of acid under both aerobiosis and anaerobiosis, whereas the addition of strain CECT 7746 to the biofilm caused a reduction only under aerobiosis.
The reduction of acid, particularly in the case of strain CECT 7747 and the supernatants thereof, is very relevant for the treatment and prevention of dental caries, since the latter is formed due to the production of acid by microorganisms when these ferment the sugars ingested in the diet. Acid pH is precisely what de-mineralises the enamel and produces caries, and, therefore, any acidogenic species, and not only Streptococci from the mutans group, could be potentially cariogenic (2). Therefore, the reduction in the production of acid is an indicator that the overall effect of treatment with the probiotic strain or the supernatant thereof is the reduction of acid and, consequently, a lower probability of developing caries.

BIBLIOGRAPHY

1. P. D. Marsh, Dental Clinics of North America 54,441 (2010).
2. P. Marsh, BMC ).
3. P. E. Petersen, Zhonghua Kou Qiang Yi Xue Za Zhi 39, 441 (Nov, 2004).
4. S. S. Socransky, A. D. et al. J Clin Periodontol 25, 134 (Feb, 1998).
5. R. P. Darveau, Nat Rev Microbiol 8, 481 (Jun 1, 2010).
6. B. J. Paster et al., J Bacteriol 183, 3770 (Jun, 2001).
7. S. R. Gill et al., Science 312, 1355 (Jun 2, 2006).
8. K. Kurokawa et al., ).
9. P. A. Vaishampayan et al., Genome Biol Evol 2010, 53 (2010).
10. J. Qin et al., Nature 464,59 ().
11. J. A. Aas et al., J Clin Microbiol 43, 5721 (Nov, 2005).
12. E. A. Grice et al., ).
13. W. J. Loesche, Microbiol Rev 50,353 (Dec, 1986).
14. M. W. Russell et al., Caries Res 38,230 (May-Jun, 2004).
15. WO 2007/077210 "Probiotic oral health promoting product".
16. WO 2005/018342 "Compositions and methods for the maintenance of oral health".
17. EP 0195672 "Dental caries preventive preparations and methods for preparing said preparations".
18. WO 2004/072093 "Antimicrobial agents".
19. Margulies M et al., ).
20. Gomez-Alvarez V et al., ISME J. 3(11) (Nov 2009).
21. Zheng Zhang et al., J Comput Biol. 7(1-2) (2000).
22. E. M. Bik et al., Isme J (Mar 25, 2010).
23. K. T. Konstantinidis et al., Proc Natl Acad Sci U S A 102, 2567 ().
24. Marchler-Bauer A et al., Nucleic Acids Res. 37:D205-10 (Jan 2009).
25. Selengut JD et al., Nucleic Acids Res. 35:D260-4 (Jan 2007).
26. Altschul et al., J Mol Bio. 215 (3):403-10 ().
27. Cole JR. et al., Nucleic Acid Res. 37:D141-5 (Jan 2009).
28. Huson DH et al., Genome Res. 17 (3):377-86 (Mar 2007).
29. Brady A and Salzberg SL. Nature Methods. 6 (9): 673-6 (Sep 2006).
30. Chen T et al., Database (Oxford) 6 (July 2010).
31. J. R. Tagg et al., Trends Biotechnol 21, 217 (May, 2003).
32. R. B. Merrifield (1963), J. Am. Chem. Soc. 85 (14): 2149-2154.
33. Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1st ed.). CRC Press. pp. 848. ISBN 0824703596.
34. Konstantinidis KT, Tiedje JM. J Bacteriol. 2005 Sep; 187(18): 6258-64.
35. Goris J et al., Int J Syst Evol Microbiol. 2007 Jan; 57(Pt 1): 81-91.
36. Richter M, Rosselló-Móra R. Proc Natl Acad Sci USA. 2009 .
37. Exterkate RA et al., Res. 2010; 44(4): 372-9.
38. Pham LC et al., Arch Oral Biol. 2011 Feb; 56(2): 136-47.

Listado de Secuencias

<110> Centro Superior de Investigación en Salud Pública (CSISP)
<120> COMPOSICIONES Y PROBIÓTICOS/PREBIÓTICOS ANTICARIES
<130> PCT-04643
<150> P201031302
<151> 2010-08-31
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 244
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (96)..(176)
<400> 1
<210> 2
<211> 666
<212> DNA
<213> Bacteria
<220>
<221> CDS
<222> (240)..(359)
<400> 2
<210> 3
<211> 42797
<212> DNA
<213> Bacteria
<400> 3
<210> 4
<211> 28023
<212> DNA
<213> Bacteria
<400> 4
<210> 5
<211> 33804
<212> DNA
<213> Bacteria
<400> 5
<210> 6
<211> 45166
<212> DNA
<213> Homo sapiens
<400> 6
<210> 7
<211> 32692
<212> DNA
<213> Homo sapiens
<400> 7
<210> 8
<211> 26
<212> PRT
<213> Homo sapiens
<400> 8
<210> 9
<211> 39
<212> PRT
<213> Bacteria
<400> 9
<210> 10
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Cebador Reverse para la obtención de fósmidos
<400> 10
ggatgtgctg caaggcgatt aagttgg 27
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Cebador Reverse para la obtención de fósmidos
<400> 11
ctcgtatgtt gtgtggaatt gtgagt 26
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Cebador T7 para la obtención de fósmidos
<400> 12
taatacgact cactataggg 20
<210> 13
<211> 34079
<212> DNA
<213> Homo sapiens
<400> 13
<210> 14
<211> 27661
<212> DNA
<213> Homo sapiens
<400> 14

Claims

Culturable anti-microbial bacterial strain selected from any of the following: CECT 7747, CECT 7746, CECT 7773 and CECT 7775.
Culturable anti-microbial bacterial strain selected from any of the following: CECT 7747, CECT 7746, CECT 7773 and CECT 7775, or a combination thereof, for use as a medicament.
Culturable anti-microbial bacterial strain selected from any of the following: CECT 7747, CECT 7746, CECT 7773 and CECT 7775, or a combination thereof, for use as an anti-microbial agent.
Culturable anti-microbial bacterial strain selected from any of the following: CECT 7747, CECT 7746, CECT 7773 and CECT 7775, or a combination thereof, for use in the prevention and/or treatment of infectious diseases of the buccal cavity.
Culturable anti-microbial bacterial strain selected from any of the following: CECT 7747, CECT 7746, CECT 7773 and CECT 7775, or a combination thereof, for use as a probiotic or functional food to improve buccal health.
Probiotic/prebiotic composition or functional food that comprises at least one anti-microbial strain according to claim 1.
Medical-pharmaceutical composition that comprises at least one anti-microbial strain according to claim 1.
Buccal health composition that comprises at least one anti-microbial strain according to claim 1.
Probiotic/prebiotic composition or functional food according to claim 6 for use in the prevention and/or treatment of infectious diseases of the buccal cavity.
Medical-pharmaceutical composition according to claim 7 for use in the prevention and/or treatment of infectious diseases of the buccal cavity.
Buccal health composition according to claim 8 for use in the prevention and/or treatment of infectious diseases of the buccal cavity.